Summary Embryonic cells polarize to develop specializations needed for morphogenesis and differentiation. Using the C. elegans embryo as a simple model, our long-term goal is to understand how early embryonic cells acquire an inner-outer polarity that regulates the cell movements of gastrulation. Many types of cells polarize when conserved PAR proteins develop asymmetric cortical localizations and regulate downstream polarity effectors. An important and largely unresolved question is how polarity cues lead to PAR asymmetry. The specific goal of this proposal is to understand how cell contacts induce an inner-outer asymmetry in PAR proteins that polarizes early embryonic cells. Our ability to combine embryological manipulations with cell biological and genetic tools provides a unique opportunity to identify and characterize these mechanisms in living embryos. Our studies should provide insights into critical contact-mediated polarity events in humans, such as the compaction of early embryonic cells required for embryonic development and the apicobasal polarization of epithelial cells needed for organogenesis. We have identified the pac-1 gene as a key regulator of inner-outer PAR asymmetry. PAC-1 contains a RhoGAP domain predicted to inhibit the activity of RHO proteins, a family of signaling molecules that includes important regulators of cell polarity. We have shown that the cortically localized RHO protein CDC- 42 is required for inner-outer PAR asymmetry and that GFP-tagged PAC-1 localizes to the inner but not outer cortex of cells. Because of these findings, here we test the hypothesis that PAC-1 controls inner- outer PAR asymmetry by spatially regulating RHO activity. Specifically, we will characterize the PAC-1 protein, determine if PAC-1 controls PAR localization by regulating the activity of RHO proteins, and test the hypothesis that two predicted interacting proteins-a-catenin and Arf small G proteins-are needed for PAC- 1 localization or function. Finally, to expand our molecular understanding of the PAC-1 polarity pathway, we will perform screens for new genes required for the asymmetric localization of PAR proteins in early embryonic cells. Together, the proposed experiments will help us build a molecular pathway from cell contacts to the asymmetric localization of PAR proteins that polarizes cells. Relevance: We are using a genetic model organism to learn how cell contacts cause embryonic cells to develop polarities essential for their function. We anticipate that our findings will help explain how cells in the human embryo develop polarities that are vital for embryonic development. [unreadable] [unreadable] [unreadable]